Nicotinic acetylcholine receptor binding of imidacloprid-related diaza compounds with various ring sizes and their insecticidal activity against Musca domestica

Article abstract of DOI:10.1002/ps.488

Fifteen 5-substituted 1-(6-chloro-3-pyridylmethyl)-2-nitromethylene-1,3-diazacyclohexanes and three other related compounds having a five- or seven-membered ring were synthesized and their biological activities were measured in vivo and in vitro. The insecticidal (in vivo) activity was evaluated against houseflies Musca domestica L under synergistic conditions with propargyl propyl phenyl phosphonate and piperonyl butoxide. The binding activity of each compound to nicotinic acetylcholine receptor in vitro was measured using [¹²⁵I]α-bungarotoxin. The insecticidal activities of the unsubstituted diazacyclohexane analogues were slightly higher than those of the imidazolidine analogues, but the enlargement of ring size to diazacycloheptane lowered the activity. Substitution of 1,3-diazacyclohexane or imidazolidine rings was not generally favourable for the activity, but the unsubstituted 1,3-diazacyclohexane analogue showed the highest binding activity. Ring substitutions and ring enlargement decreased the activity 100-30000-fold.

Full text of DOI:10.1002/ps.488

Pest Management Science
Pest Manag Sci 58:483±490 online: 2002)
DOI: 10.1002/ps.488
Nicotinic acetylcholine receptor binding of
imidacloprid-related diaza compounds with
various ring sizes and their insecticidal activity
against Musca domestica
Shinzo Kagabu,1* Hisashi Nishiwaki,2 Kazuyuki Sato,2 Manabu Hibi,1
Nahato Yamaoka1 and Yoshiaki Nakagawa2
1Department of Chemistry, Faculty of Education, Gifu University, Gifu 501-1193, Japan
2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Abstract: Fifteen 5-substituted 1-ꢀ6-chloro-3-pyridylmethyl)-2-nitromethylene-1,3-diazacyclohex-
anes and three other related compounds having a ®ve- or seven-membered ring were synthesized
and their biological activities were measured in vivo and in vitro. The insecticidal ꢀin vivo) activity was
evaluated against house¯ies Musca domestica L under synergistic conditions with propargyl propyl
phenyl phosphonate and piperonyl butoxide. The binding activity of each compound to nicotinic
acetylcholine receptor in vitro was measured using [125I]a-bungarotoxin. The insecticidal activities of
the unsubstituted diazacyclohexane analogues were slightly higher than those of the imidazolidine
analogues, but the enlargement of ring size to diazacycloheptane lowered the activity. Substitution of
1,3-diazacyclohexane or imidazolidine rings was not generally favourable for the activity, but the
unsubstituted 1,3-diazacyclohexane analogue showed the highest binding activity. Ring substitutions
and ring enlargement decreased the activity 100±30000-fold.
# 2002 Society of Chemical Industry
Keywords: insecticidal activity; Musca domestica; chloronicotinyl insecticides; neonicotinoids; [125I]a-bungaro-
toxin; nicotinic acetylcholine receptor; 2-nitromethylene-1,3-diazacyclohexane
1
INTRODUCTION
the reference in the 3-D lattice. A charge ‡1) and an
sp3 carbon atom are placed at the various intersections
of the 3-D lattice and electrostatic and steric interac-
tions are calculated for all atoms, and the optimum
latent variables are extracted by the partial least-square
method of Wold et al. 23 In CoMFA the electrostati-
cally and sterically favourable/unfavourable ®elds
surrounding the molecules are determined, and may
be helpful in drug design. In our compound set,
various structures are included: 1) NNO2 at the 2-
position of the imidazolidine ring is substituted with
CHNO2, NCN or CHCN, 2) the N atom at the 3-
position is replaced with S, O or C, 3) the 6-chloro-3-
pyridylmethyl moiety is replaced with various struc-
tures such as benzyl, 6-chloro-3-pyridylethyl, etc.17 In
a further study we have combined nitenpyram
derivatives which have acyclic structure in the imida-
zolidine moiety.3,18 The latest CoMFA gave the
favourable and unfavourable steric/electrostatic ®elds
essential for the binding activity, but the regions
surrounding the pyridine ring and the C4-C5 and N3
positions of imidazolidine ring of imidacloprid were
not clearly described because the structural variations
are small with respect to these moieties.17
Imidacloprid and related chloronicotinyl compounds
or neonicotinoids) have distinctive chemical struc-
tures, and these compounds are insecticidally very
potent against various pest species.1±7 It has been
shown that these compounds have agonistic effects on
insect nicotinic acetylcholine receptor nAChR) in
various bioassay systems.8±10 The correlation analyses
between insecticidal activity and the binding activity to
nAChR for a set of compounds clearly indicated that
the insecticidal action of these compounds is caused by
their binding to nAChR of insects.11±13 Structure±
activity relationship analyses have also been performed
for the in vitro activities.9,11±20 In particular, three-
dimensional quantitative structure±activity relation-
ship 3-D QSAR) procedures are helpful to predict the
receptor±ligand interactions.17,18,21
We have applied comparative molecular ®eld
analysis CoMFA), a 3-D QSAR procedure,22 to the
binding activity of imidacloprid-related com-
pounds.17,18 In CoMFA each active structure of a
compound is superposed onto that of a reference
compound so that each structural component is as
close as possible to the corresponding component of
* Correspondence to: Shinzo Kagabu, Department of Chemistry, Faculty of Education, Gifu University, Gifu 401-1193, Japan
E-mail: kagabus@cc.gifu-u.ac.jp
(Received 5 October 2001; revised version received 2 January 2002; accepted 30 January 2002)
# 2002 Society of Chemical Industry. Pest Manag Sci 1526±498X/2002/$30.00
483

S Kagabu et al
To clarify these uncertain ®elds we have synthesized
a series of substituted benzyl derivatives,13 N3-
alkylated imidacloprid analogues14,19 and correspond-
ing CHNO2 derivatives.14,24 In this paper we report
the synthesis of a new series of compounds including
1,3-diazacyclohexane 6-membered ring) and 1,3-
diazacycloheptane 7-membered ring) as well as 4-
or 5-substituted imidazolidine analogues. We also
introduced various substituents at the 5-position of the
1,3-diazacyclohexane ring. The insecticidal activity
and the binding activity against house¯ies were
measured and their relationship to structure was
analyzed.
anhydrous magnesium sulfate. Evaporation of the
solvent afforded 1.97g of N- 6-chloro-3-pyridyl-
methyl)-2-n-butyl-1,3-propanediamine in 84% crude
yield. A mixture of 1.94g 7.6mmol) of crude diamine
and
1,1-bis methylthio)-2-nitroethylene
1.25g,
7.6mmol) in 30ml of ethanol was heated under re¯ux
for 27h. After evaporation of the ethanol, the semi-
solid residue was dissolved in hot ethyl acetate after
adding a small amount of activated charcoal, and
®ltered while hot. Colourless needles of 7 crystallized
from the ®ltrate while standing at room temperature.
The analytical sample was recrystallized twice from
ethanol. The yield was 0.65g 26%).
2.2.2 1-ꢀ6-Chloro-3-pyridylmethyl)-4,4-dimethyl-2-
nitromethyleneimidazolidine ꢀ17)
2
MATERIALS AND METHODS
2.1 Chemicals
A solution of 1,2-diamino-2-methylpropane 1.90g,
22mmol) in acetonitrile 10ml) was added dropwise
to a solution of 6-chloro-3-pyridylmethyl chloride
0.70g, 4.3mmol) in acetonitrile 10ml) and then
the resulting solution was re¯uxed overnight. After
removing the insoluble solids by ®ltration, the ®ltrate
was evaporated in vacuum. Distilled water was added
to the residue, which was then extracted with di-
chloromethane. The separated organic layer was dried
over anhydrous magnesium sulfate. Evaporation of the
The compounds used in this study were newly
synthesized by the published methods.24,25 Represen-
tative procedures are described here for compounds 7,
17 and 18 see Fig 1). All melting points are uncor-
rected. NMR spectra were obtained by a Varian
Gemini 2000 C/H instrument 400MHz). The
chemical shifts were recorded in d ppm) and the
coupling constants in Hz. Mass spectra were recorded
EI, 70eV) with a Shimadzu QP 1000 mass spectro-
meter. Log P values of newly synthesized compounds
were measured in 1-octanol‡water using the ¯ask-
shake method. a-Bungarotoxin a-BGTX) was pur-
chased from Sigma Chemical Co St Louis, MI,
USA), and [125I]a-BGTX was obtained from Amer-
sham Pharmacia Biotech Buckinghamshire, UK).
Propargyl propyl phenyl phosphonate NIA16 388 or
NIA) was obtained from our stock sample as prepared
by Nakagawa et al. 26 Piperonyl butoxide PB), an
inhibitor of mixed function oxidases, was purchased
from Nacalai Tesque Kyoto, Japan).
solvent afforded
a
mixture of N- 6-chloro-3-
pyridylmethyl)-1,1-dimethylethylenediamine and N-
6-chloro-3-pyridylmethyl)-2,2-dimethylethylenedi-
amine 0.98g, quantitative yield). A solution of the
mixture 0.92g, 4.3mmol) in ethanol 20ml) was
added dropwise to a suspension of 1,1-bis methyl-
thio)-2-nitroethylene 0.69g, 4.2mmol) and potas-
sium carbonate 0.57g, 4.2mmol) in ethanol 20ml),
and the resulting mixture was re¯uxed overnight. After
®ltration of the precipitates, the ®ltrate was concen-
trated. The residue was recrystallized from ethanol to
afford the product 17. The yield was 0.23g 19%).
2.2 Synthesis
2.2.1 5-n-Butyl-1-ꢀ6-chloro-3-pyridylmethyl)-2-
nitromethylene-1,3-diazacyclohexane ꢀ7)
2.2.3 1-ꢀ6-Chloro-3-pyridylmethyl)-5,5-dimethyl-2-
nitromethyleneimidazolidine ꢀ18)
6-Chloronicotinaldehyde 1.30g, 9.21mmol) was
added in small portions to a re¯uxing solution of
2-butyl-1,3-propanediamine 1.49g, 11.5mmol) in
benzene 30ml). Heating was continued until no
water separated in an attached Dean±Stark trap
2ꢀ3h). The benzene was distilled off under reduced
pressure and the remaining imine was used without
further puri®cation. Powdered NaBH4 1.47g,
38.8mmol) was added in small portions to a suspen-
sion of the imine in ethanol‡water 5‡1 by volume).
The resulting mixture was stirred at room temperature
for 24 h. After most of the ethanol had been distilled
off under a weak vacuum, the diamine was extracted
with isopropyl ether IPE) from the water phase.
Evaporation of IPE left an oily liquid, which was taken
up with hydrochloric acid 1M) and washed with IPE
2Â10ml). The aqueous solution was alkalized to pH
12 with solid sodium hydroxide with ice cooling,
extracted with chloroform 5Â10ml), and dried over
The ®ltered solution remaining after recrystallization
of 17 was concentrated. The residue was subjected to
silica gel column chromatography with ethanol‡ethyl
acetate 1‡2 by volume) as eluate. The collected
crude product was developed on HPLC Shimadzu,
LC-6A; COSMOSIL 5C18-AR, 20Â250mm) with
water‡methanol 65‡35 by volume) as mobile
phase. An analytical pure sample was obtained from
the fractions at a retention time of 24.2min. The yield
was 0.07g 5%).
2.3 Bioassay
2.3.1 Insecticidal activity
The method for the insecticidal test against house¯ies
was that previously reported.15 In brief, a methanol
solution 1ml) containing NIA 0.2%) and PB 0.2%)
was topically applied to the abdomen of female
house¯ies anaesthetized using carbon dioxide. After
1h at 25°C, solutions 0.24ml) of test compound in
484
Pest Manag Sci 58:483±490 online: 2002)

Insecticidal and binding activities of imidacloprid analogues
ethanol‡water 50‡50 by volume) at various con-
centrations were injected into the dorsal side of the
thorax. After 1h at 25°C, the number of dead and
paralyzed ¯ies was counted. The 50% effective
concentration, EC50 M), was evaluated from the
calculated by
Ki ˆ IC50=ꢁ1 ‡ ‰LŠ=Kd†;
where [L] is the concentration of [125I]a-BGTX and
Kd is the dissociation constant of a-BGTX. The Kd
value of a-BGTX was determined in each membrane
preparation. The mean Kd of a-BGTX binding to
membrane preparations used to estimate the Ki value
of test compounds was 0.31 Æ0.15) nM n =4). This
Kd value was consistent with the value 0.29nM) that
we previously estimated.14 The reciprocal logarithm of
the Ki M) value, pKi, was calculated for each com-
pound.
dose±response
relationship
using
probit
methods.27,28 The reciprocal logarithm of the EC50
value, pEC50, was de®ned as the insecticidal activity.
2.3.2 Binding activity
The procedure for the receptor binding assay was that
previously reported.13,17 Brie¯y, the membrane pre-
paration obtained from house¯y heads was incubated
with test compounds and [125I]a-BGTX 0.2nM) at
24°C for 60min. The reaction was terminated by
rapid ®ltration through a Uni®lter GF/B Packard
Instrument Co, Meriden, CT, USA), which had been
treated with polyethyleneimine 0.1%). The ®lters
were rinsed three times with sodium phosphate
10mM; pH 7.4) containing sodium chloride
50mM), followed by methanol. After adding Micro-
scinti-O Packard Instrument Co, Meriden, CT,
USA) as a scintillation cocktail, the radioactivity was
measured using a Topcount instrument Packard
Instrument Co, Meriden, CT, USA). The molar con-
centration required for 50% inhibition of the speci®c
binding of [125I]a-BGTX, IC50 M), was determined
by a non-linear regression analysis using PRISM
Graphpad Software Inc, San Diego, CA, USA).
The binding activity was expressed as the Ki M) value
3
RESULTS
3.1 Chemistry
The structures of test compounds are shown in Fig 1.
The NMR assignments of the protons and the main
MS fragments of the newly prepared compounds are
given in Tables 1 and 2, respectively. The physical and
spectral data for the prepared compounds are given in
Tables 1 and 2. The new compounds gave satisfactory
elemental analyses forC, H and N. Log P values of the
compounds range from À0.80 to 1.55 and are listed in
Table 3.
3.2 Insecticidal activity
The activity values varied from 3.99 compound 14) to
6.12 compound 1) as listed in Table 3, showing an
activity change of more than 100-fold as a result of
imidazolidine ring modi®cations. Notably, the unsub-
Figure 1. Imidacloprid and related
chloronicotinyl compounds.
Pest Manag Sci 58:483±490 online: 2002)
485

S Kagabu et al
Table 1. 1H spectral data of newly prepared compoundsa
Chemical shifts ꢀppm)
Compound
no
NCH2CꢀXY)CH2N b
3.44 ꢀm), 3.50 ꢀm)
CHX
CH2
=CHNO2
Pyridine and others
1
2.11 ꢀm) 4.43
6.23
7.37 ꢀd, J=8.4), 7.53 ꢀdd, J=8.4/2.6),
8.25 ꢀd, J=2.6), 10.82 ꢀNH, bs)
1.07 ꢀ3H, d, J=6.6), 7.36 ꢀd, J=8.0),
7.54 ꢀdd, J=8.0/2.5), 8.25 ꢀd, J=2.5),
10.8 ꢀNH, bs)
2
3
4
5
6
7
8
3.05 ꢀ2H, m)
3.32 ꢀ1H, m)
3.45 ꢀ1H, m)
3.06 ꢀs), 3.17 ꢀs)
2.20 ꢀm) 4.40 ꢀd, J=17.2),
4.46 ꢀd, J=17.2)
6.64
6.71
6.62
6.63
6.61
6.63
6.64
4.43
1.98 ꢀm) 4.45
2.06 ꢀm) 4.46
1.08 ꢀ6H, s), 7.38 ꢀd, =7.7)
7.57 ꢀdd, J=7.7/1.8), 8.29 ꢀd, J=1.8)
10.9 ꢀNH, bs)
0.96 ꢀ3H, t, J=7.7), 1.98 ꢀ2H, m),
7.36 ꢀd, J=8.1), 7.54 ꢀdd, J=8.1/2.2),
8.24 ꢀd, J=2.2), 10.8 ꢀNH, bs)
0.92 ꢀ3H, m), 1.34 ꢀ4H, m), 7.36 ꢀd, J=8.1),
7.54 ꢀdd, J=8.1/1.8), 8.24 ꢀd, J=1.8),
10.8 ꢀNH, bs)
0.94 ꢀ3H, d, J=6.6), 0.98 ꢀ3H, d, J=8.0),
1.58 ꢀ1H,m), 7.35 ꢀd, J=8.0),
7.53 ꢀdd, J=8.0/2.6), 10.8 ꢀNH, bs)
0.89 ꢀ3H, t, J=6.6), 1.31 ꢀ6H, m),
7.36 ꢀd, J=8.4), 7.54 ꢀdd, J=8.4/2.6),
8.24 ꢀd, J=2.6), 10.8 ꢀNH, bs)
0.91 ꢀ6H, d, J=6.6), 1.20 ꢀ2H, m),
1.58 ꢀ1H, m), 7.37 ꢀd, J=8.0),
7.53 ꢀdd, J=8.0, 2.6), 8.25 ꢀd, J=2.6),
10.8 ꢀNH, bs)
3.10 ꢀ2H, m)
3.36 ꢀ1H, m)
3.58 ꢀ1H, m)
3.08 ꢀ2H, m),
3.34 ꢀ1H, m),
3.57 ꢀ1H, m)
3.16 ꢀ2H, m),
1.81 ꢀm) 4.43 ꢀd, J=17.2)
4.48 ꢀd, J=17.2)
3.35 ꢀ1H, ddd, J=12.5/4.6/2.2),
3.60 ꢀ1H, m, J=13.2)
3.08 ꢀ2H, m),
3.35 ꢀ1H, m),
3.57 ꢀ1H, m)
2.05 ꢀm) 4.44
2.14 ꢀm) 4.43
3.06 ꢀ2H, m), 3.31 ꢀ1H, m),
3.56 ꢀ1H, m)
9
3.18 ꢀ2H, m), 3.31 ꢀ1H, m),
3.57 ꢀ1H, m)
1.94 ꢀm) 4.41 ꢀd, J=17.2),
4.47 ꢀd, J=17.2)
6.62
0.86±0.95 ꢀ3H‡3H, m), 1.22 ꢀ1H,m),
1.44 ꢀ2H, m), 7.37 ꢀd, J=8.1),
7.52 ꢀdd, J=8.1/2.2), 8.24 ꢀd, J=2.2),
10.8 ꢀNH, bs)
10
11
12
13c
3.13±3.29 ꢀ3H, m),
3.57 ꢀ1H, m)
1.85 ꢀm) 4.41 ꢀd, J=17.2),
4.47 ꢀd, J=17.2)
6.61
6.70
6.64
6.51
0.95 ꢀ9H, s), 7.37 ꢀd, J=8.4),
7.51 ꢀdd, J=8.4/2.6), 8.24 ꢀd, J=2.6),
10.8 ꢀNH, bs)
7.20 ꢀC6H5, 2H, m), 7.30±7.39 ꢀC6H5, 4H, m)
7.48 ꢀdd, J=8.1/2.6), 8.23 ꢀd, J=2.6),
11.0 ꢀNH, bs)
3.46±3.61 ꢀ3H, m),
3.74 ꢀ1H, m)
3.30 ꢀm) 4.47
2.39 ꢀm) 4.39
3.16 ꢀ2H, m),
3.30 ꢀ1H,,m),
3.50 ꢀ1H, m)
2.61±2.73 ꢀC6H5CH2, m), 7.50 ꢀdd, J=8.4/2.5
8.21 ꢀd, J=2.5), 10.8 ꢀNH, bs)
3.25 ꢀ1H, d, J=13.2),
3.34 ꢀ1H, d, J=12.1),
3.45 ꢀ1H, d, J=12.1),
3.62 ꢀ1H, d, J=13.2)
3.40 ꢀ1H, d, J=14.2),
3.45 ꢀ1H, d, J=14.2)
3.63 ꢀ1H, d, J=12.1)
3.66 ꢀ1H, d, J=12.1)
3.55 ꢀ1H, d, J=13.9),
3.62 ꢀ1H, d, J=13.9),
3.71 ꢀ1H, d, J=13.2),
3.86 ꢀ1H, d, J=13.2)
3.59 ꢀ2H, m), 3.81 ꢀ2H, m)
4.14 ꢀm) 4.55 ꢀd, J=18.0),
4.64 ꢀd, J=18.0)
5.50 ꢀOH, d, J=3.2), 7.54 ꢀd, J=8.5),
7.80 ꢀdd, J=8.5/2.2),
8.39 ꢀd, J=2.2), 10.4 ꢀNH, bs)
14c
15c
3.85 ꢀm) 4.56 ꢀd, J=17.6),
4.65 ꢀd, J=17.6)
6.52
6.61
3.29 ꢀOCH3), 7.55 ꢀd, J=8.4),
7.75 ꢀdd, J=8.4/2.2), 8.33 ꢀd, J=2.2),
10.4 ꢀNH, bs)
5.07 ꢀm) 4.55 ꢀd, J=17.6),
4.70 ꢀd, J=17.6)
6.95±7.01 ꢀC6H5, 3H,m),
7.31 ꢀC6H5, dd, J=8.3/8.4), 7.52 ꢀd, J=8.4),
7.76 ꢀdd, J=8.4/2.6), 8.36 ꢀd, J=2.6),
10.5ꢀNH)
7.37 ꢀ1H, d, J=8.1), 7.59 ꢀ1H, dd, J=8.1/2.5)
8.31 ꢀ1H, d, J=1.8), 8.73 ꢀNH, bs)
1.42 ꢀ6H, s), 7.38 ꢀ1H, d, J= 8.1),
7.55 ꢀ1H, dd, J= 8.4 /2.4),
8.28 ꢀ1H, d, J= 2.4), 8.70 ꢀNH, bs)
1.37 ꢀ6H, s), 7.34 ꢀ1H, d, J= 8.1),
7.54 ꢀ1H, dd, J= 8.4/2.4),
16c
17
4.33
4.29
6.66
6.61
3.28 ꢀ2H, s)
3.62 ꢀ2H, s)
18
19
4.49
4.41
6.33
6.52
8.30 ꢀ1H, d, J= 2.4), 8.71 ꢀNH, bs)
7.37 ꢀ1H, d, J=8.1), 7.55 ꢀ1H, dd, J=8.4/2.4)
8.30 ꢀ1H, d, J=2.4), 10.06 ꢀ1H, NH, bs)
1.63 ꢀ2H, m), 1.75 ꢀ2H, m),
3.26 ꢀ2H, m),
3.48 ꢀ2H, dd, J=10.5/5.0)
a In deuterochloroform unless otherwise stated; coupling constant J ꢀHz) for H±H.
b CH2CH2 for compound 16, CH2CꢀCH3)2 for compound 17, CꢀCH3)2CH2 for compound 18, CH2CH2CH2 for compound 19.
c In hexadeuterodimethyl sulfoxide.
486
Pest Manag Sci 58:483±490 online: 2002)

Insecticidal and binding activities of imidacloprid analogues
Table 2. Melting points and mass spectra of newly prepared compounds
Compound Mp
no
ꢀ°C)
m/z ꢀIntensity)
‡
‡
‡
‡
‡
‡
‡
‡
1
2
3
4
5
6
7
8
9
186 268 ꢀM , 22%), 223 ꢀ39%), 222 ꢀ76%), 128 ꢀ40%), 126 ꢀ100%)
198 282 ꢀM , 15%), 252 ꢀ11%), 236 ꢀ100%), 200 ꢀ19%), 195 ꢀ14%), 126 ꢀ73%)
223 296 ꢀM , 6%), 250 ꢀ81%), 214 ꢀ11%), 126 ꢀ96%), 41 ꢀ100%)
167 296 ꢀM , 8%), 250 ꢀ100%), 214 ꢀ18%), 126 ꢀ88%)
172 310 ꢀM , 3%), 264 ꢀ78%), 222 ꢀ16%), 126 ꢀ89%), 44 ꢀ100%)
197 310 ꢀM , 9%), 264 ꢀ100%), 228 ꢀ26%), 223 ꢀ22%), 126 ꢀ100%)
146 324 ꢀM , 2%), 278 ꢀ70%), 237 ꢀ16%), 126 ꢀ100%)
203 324 ꢀM , 4%), 294 ꢀ11%), 278 ꢀ100%), 238 ꢀ16%), 223 ꢀ29%), 127 ꢀ17%)
‡
‡
190 324 ꢀM , 2%), 278ꢀ37%), 234 ꢀ36%), 223 ꢀ17%), 157 ꢀ12%), 127 ꢀ100%)
10
221 324 ꢀM , 17%), 295 ꢀ28%), 290 ꢀ28%), 278 ꢀ100%), 234 ꢀ83%), 223 ꢀ42%), 209 ꢀ69%), 157 ꢀ43%), 141 ꢀ37%),
127 ꢀ48%)
‡
11
12
13
14
15
16
17
18
19
218 344ꢀM , 2%), 298 ꢀ82%), 126 ꢀ51%), 104 ꢀ100%)
‡
210 344 ꢀM , 14%), 312 ꢀ41%), 233 ꢀ24%), 127 ꢀ77%), 118 ꢀ62%), 91 ꢀ100%)
‡
205 284 ꢀM , 2%), 238 ꢀ33%), 209 ꢀ15%), 155 ꢀ13%), 126 ꢀ100%)
‡
207 299 ꢀM ‡1, 48%), 268 ꢀ16%), 252 ꢀ76%), 222 ꢀ14%), 126 ꢀ86%)
‡
‡
‡
‡
202 360 ꢀM ‡1, 8%), 314 ꢀ24%), 267 ꢀ12%), 220 ꢀ13%), 126 ꢀ100%), 108 ꢀ11%)
167 254 ꢀM , 25%), 208 ꢀ70%), 172 ꢀ40%), 126 ꢀ100%)
195 282 ꢀM , 16%), 267 ꢀ24%), 252 ꢀ17%), 236 ꢀ80%), 126 ꢀ92%), 55 ꢀ100%)
208 282 ꢀM , 15%), 267 ꢀ15%), 252 ꢀ13%), 236 ꢀ89%), 126 ꢀ94%), 55 ꢀ100%)
‡
219 282 ꢀM , 3%), 236 ꢀ79%), 126 ꢀ88%), 55 ꢀ100%)
stituted six-membered ring compound 1) was more
potent than either the ®ve-membered ring homologue
16) or imidacloprid 20). Substitution with isopropyl
6) or n-butyl 7) at the 5-position of the diazacyclo-
hexyl ring did not reduce activity. However, other
straight or branched alkyl groups 2±5, 8±10), as well
as phenyl 11) and benzyl 12) groups decreased the
activity substantially. An alcohol 13) or ethers 14, 15)
were less potent than unsubstituted compound 1) by
a factor of 100. The seven-membered ring homologue
19) exhibited slightly lower potency than the un-
substituted compound 1). Also the activity of the
imidazolidine compound 16) was decreased by
geminally appending two methyl groups at the 4-
17) or 5-position 18), although compound 18 was
three times more active than compound 17. We could
not ®nd any de®nitive relationship between the
insecticidal activity and the molecular hydrophobicity
log P.
correlation between the binding activity and log P was
observed.
4
DISCUSSION
The CoMFA maps predicted the permissible steric
®eld area in the area extending from the imidazolidine
ring.17,18 The slight enhancement of the binding
activity by enlarging the ring from ®ve-membered
16) to six-membered 1) is not inconsistent with this
CoMFA result. However, introduction of substituents
at the 5-position of diazacyclohexane as well as the
introduction of methyl groups at either the 4- or 5-
position of the imidazolidine ring were fairly unfavour-
able to activity, suggesting the existence of a sterically
unfavourable ®eld over this permissible ®eld. CoMFA
is in progress for the combined set of compounds.
As a whole, however, the relationships between
substituents and biological potencies appear neither
uniform nor simple. As for the insecticidal activity, all
substituents tested excepting isopropyl 6) and n-butyl
7) decreased the potency of the parent molecule 1 by
different scalars. It is puzzling why the sec-butyl
derivative 9 exhibited 6±70 times higher potency than
the other butyl isomers with respect to the binding
activity. To uncover the structural features required
for the enhancement of the biological activity a
quantitative analysis is important.
3.3 Binding activity
The binding activity of compound 1 was twice that of
the ®ve-membered ring homologue 16), and it was
about 700-fold more active than imidacloprid 20).
The activity was decreased by further enlarging to a
seven-membered ring 19). The activity was also
lowered drastically by putting alkyl, phenyl, hydroxyl
and aralkyloxy groups at the 5-position of the
diazacyclohexyl ring, with the exception of sec-butyl
9). Compound 9 showed the highest binding potency
among the diazacyclohexyl derivatives. Another point
to note is that the potencies were different by over 15
times in the regional isomers 17 and 18. Here again no
Previously we reported that insecticidal activity was
positively correlated with the binding activity for the
substituted benzyl derivatives of chloronicotinyl in-
Pest Manag Sci 58:483±490 online: 2002)
487

S Kagabu et al
Table 3. Binding and insecticidal activities of chloronicotinyl compounds with modified imidazolidine ring
Compound
no
Hydrophobicity
RInsecticidal PEC
50 ꢀM) a Binding PKi ꢀM) a
log P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H
6.12 ꢀÆ0.12) ꢀ2)
8.28 ꢀÆ0.14) ꢀ2)
5.38 ꢀÆ0.27) ꢀ7)
3.98 ꢀÆ0.28) ꢀ5)
4.98 ꢀÆ0.06) ꢀ2)
5.06 ꢀÆ0.13) ꢀ2)
5.43 ꢀÆ0.02) ꢀ2)
5.29 ꢀÆ0.13) ꢀ2)
5.04 ꢀÆ0.17) ꢀ2)
6.04 ꢀÆ0.12) ꢀ2)
4.19 ꢀÆ0.16) ꢀ2)
3.62 ꢀÆ0.29) ꢀ2)
5.01 ꢀÆ0.07) ꢀ2)
3.69 ꢀÆ0.16) ꢀ2)
3.62 ꢀÆ0.23) ꢀ2)
3.98 ꢀÆ0.20) ꢀ2)
7.93c
À0.30
À0.10
0.40
CH3
di-CH3
C2H5
4.60 ꢀÆ0.17) ꢀ3)
4.57 ꢀÆ0.13) ꢀ2)
5.34 ꢀÆ0.13) ꢀ2)
4.80 ꢀÆ0.18) ꢀ2)
6.01 ꢀÆ0.28) ꢀ3)
6.10 ꢀÆ0.09) ꢀ2)
4.84 ꢀÆ0.07) ꢀ2)
4.44 ꢀÆ0.08) ꢀ2)
4.72 ꢀÆ0.18) ꢀ2)
4.70 ꢀÆ0.12) ꢀ2)
4.19 ꢀ1)
0.50
n-C3H7
i-C3H7
n-C4H9
i-C4H9
s-C4H9
t-C4H9
C6H5
1.07
0.91
1.55
1.46
1.40
1.25
1.22
C6H5CH2
OH
1.44
4.30 ꢀÆ0.13) ꢀ2)
3.99 ꢀ1)
À0.80
À0.42
1.13
À0.19d
CH3O
C6H5O
4.20 ꢀÆ0.13) ꢀ2)
5.80b
17
18
19
5.01 ꢀÆ0.05) ꢀ2)
5.54 ꢀÆ0.06) ꢀ2)
3.48 ꢀÆ0.29) ꢀ3)
4.65 ꢀÆ0.02) ꢀ3)
0.37
0.31
5.70 ꢀÆ0.21) ꢀ2)
6.01 ꢀÆ0.27) ꢀ2)
0.34
20
Imidacloprid
5.71b
5.43c
0.58d
a
Mean values with standard deviation with the number of replications in parentheses.
b
c
d
Cited from Reference 15.
Cited from Reference 12.
Cited from Reference 8.
secticides [eqn 1)].15
structural feature of the N3-substituents [eqn 2)].14
pEC50 ˆ0:611ꢁÆ0:132† pKi À 0:923 ꢁÆ0:303† ICHNO
pEC50 ˆ 0:688 ꢁÆ0:197† pKi ‡ 0:395 ꢁÆ1:071† ꢁ1†
n ˆ 16; r ˆ 0:894; s ˆ 0:439; F1;14 ˆ 55:752
2
À 0:795 ꢁÆ0:349† Ibranch=N3 ‡ 1:909 ꢁÆ0:485† ꢁ2†
n ˆ 26; r ˆ 0:929; s ˆ 0:303; F3;22 ˆ 46:101
In eqn 1) and the following equations, n is the number
of compounds, s is the standard deviation, r is the
correlation coef®cient, and F is the value of the ratio
between regression and residual variances. The ®gures
in parentheses following each coef®cient are the 95%
con®dence intervals of the regression coef®cient. We
also found a high correlation between these two
activities for N3-substituted imidacloprid derivatives
after taking into account the structural difference of
the nitroimino and nitromethylene moieties and a
In eqn 2), the ICHNO2 term was set as 1 for compounds
having nitromethylene group instead of nitroimino
group in the imidazolidine moiety of imidacloprid.
The Ibranch/N3 term =1) was given for the compounds
to which a secondary alkyl or phenyl group was
introduced. Since the coef®cients of the pKi terms
were similar between eqns 1) and 2), these two sets
488
Pest Manag Sci 58:483±490 online: 2002)

Insecticidal and binding activities of imidacloprid analogues
cyclohexyl, diazacycloheptyl and dimethylimidazoli-
dine derivatives are about 20-fold higher than the
predicted values from their binding activity. The
reason why Imod was insigni®cant for compound 1 is
probably due to the bulkiness of 1,3-diazacyclohexyl
ring being close to that of the imidazolidine ring, but
not as large as that for alkylated 1,3-diazacyclohexyl or
1,3-diazacycloheptyl rings.
We have examined how hydrophobicity participates
in the regression analysis for chloronicotinyl-related
compounds in previous papers.9,17,19,20 The correla-
tion between the insecticidal and neurophysiological
activities was improved by adding the negative log P)2
term.9,19,20 The relationship between the binding and
neurophysiological activities was also improved by
including the log P term with a positive sign.17 Since
the signs of the hydrophobicity parameter log P) are
opposite in these correlation equations insecticidal±
neurophysiological and neurophysiological±binding),
the hydrophobicity parameter could be cancelled in
the correlation between insecticidal and binding
activities, as previously discussed.14 In fact, the
addition of log P in eqn 4) was insigni®cant.
To date we have discussed the QSAR based on the
binding potencies using labelled a-BGTX. However,
recently it has been suggested that the binding assay is
more straightforward when imidacloprid is used
instead of a-BGTX as radioligand.11,29 There is also
an interesting argument that neonicotinoids possibly
bind to distinct sites from a-BGTX, although on the
same receptor, or on different receptors.30±33 We
recently measured the activity of a set of compounds in
a binding assay using [3H]imidacloprid and obtained a
similar structure±activity relationship for a set of com-
pounds. However, further considerations are required
for a structure±activity relationship study of the wide
rage of structures as suggested by Wollweber and
Tietjen.34 We are continuing our research to elucidate
the binding mode for neonicotinoids, including
chloronicotinyl compounds, by comparing the corre-
lation equations using both ligands.
Figure 2. Plot of observed insecticidal activity vs calculated values from
eqn (3). Newly measured compounds were shown by closed circles and
triangles. Open circles are cited from our previous publications (References
13–15). Numbers in figure corresponds to the compound number in Fig 1
and Tables. Compounds 9 and 12 specified by closed triangles were not
used to derive eqn (4).
of compounds were combined to formulate eqn 3):
pEC50 ˆ0:633 ꢁÆ0:120† pKi À1:065 ꢁÆ0:303† ICHNO
2
À 0:719 ꢁÆ0:402† Ibranch=N3 ‡ 1:824 ꢁÆ0:460† ꢁ3†
n ˆ 41; r ˆ 0:894; s ˆ 0:369; F3;37 ˆ 48:935
We calculated the insecticidal activity in terms of
pEC50 of the present set of the compounds by eqn 3),
and found that the observed pEC50 values were
signi®cantly higher than the calculated ones except
for compounds 9 and 12 as shown in Fig 2. Thus,
another indicator variable Imod which takes 1 for the
substituted diazacyclohexyl, dimethylimidazolidine
and diazacycloheptyl compounds was considered to
derive eqn 4) for all sets of compounds excluding
compounds 1 H), 9 sec-Bu), and 12 CH2C6H5). For
unsubstituted diazacyclohexyl compounds, Imod is set
as 0, because the activity is well predicted by eqn 3),
as shown in Fig 2.
ACKNOWLEDGEMENTS
We are grateful to Dr Everett Bandman of the Uni-
versity of California Davis for reviewing this manu-
script. A part of this study was performed in the RI
Centre of Kyoto University.
pEC50 ˆ0:626 ꢁÆ0:104† pKi À 1:049 ꢁÆ0:309† ICHNO
2
À 0:726 ꢁÆ0:427† Ibranch=N3
‡ 1:307 ꢁÆ0:265† Imod ‡ 1:850 ꢁÆ0:419†
ꢁ4†
REFERENCES
1 Kagabu S, Chloronicotinyl insecticides-discovery, application
and future perspective. Rev Toxicol 1:75±129 1997).
2 Elbert A, Nauen R and Leicht W, Imidacloprid, a novel
chloronicotinyl insecticide: biological activity and agricultural
importance, in Insecticides with novel modes of action, mechanism
and application, ed by Ishaaya I and Degheele D, Springer,
Berlin, pp 50±73 1998).
n ˆ 57; r ˆ 0:912; s ˆ 0:397; F4;52 ˆ 64:131
Coef®cients of pKi, ICHNO , and Ibranch/N3, in eqn 4)
2
are consistent with those in eqn 3). Equation 4)
means that the insecticidal activity correlates linearly
with the binding activity for all compounds. The new
indicator variable Imod term in eqn 4) shows that the
insecticidal activity values of the substituted diaza-
3 Akayama A and Minamida I, Discovery of a new systemic
insecticide, nitenpyram and its insecticidal properties, in
Nicotinoid insecticides and the nicotinic acetylcholine receptor, ed
Pest Manag Sci 58:483±490 online: 2002)
489

S Kagabu et al
by Yamamoto I and Casida JE, Springer, Berlin, pp 127±148
H, Nishimura K and Ueno T, Three-dimensional quantitative
structure±activity relationship analysis of acyclic and cyclic
chloronicotinyl insecticides. Pestic Manag Sci 56:509±515
2000).
1999).
4 Yamada T, Takahashi H and Hatano R, A novel insecticide,
acetamiprid, in Nicotinoid insecticides and the nicotinic acetyl-
choline receptor, ed by Yamamoto I and Casida JE, Springer,
Berlin, pp 149±176 1999).
19 Nishimura K, Tanaka M, Iwaya K and Kagabu S, Relationship
between insecticidal and nerve-excitatory activities of imida-
cloprid and its alkylated congeners at the imidazolidine NH
site. Pestic Biochem Physiol 62:172±178 1998).
5 Maien®sch P, Brandl F, Kobel W, Rindlisbacher A and Senn R,
CGA 293343: a novel, broad-spectrum neonicotinoid insecti-
cide, in Nicotinoid insecticides and the nicotinic acetylcholine
receptor, ed by Yamamoto I and Casida JE, Springer, Berlin, pp
177±209 1999).
20 Kiriyama K, Iwaya K, Kagabu S and Nishimura K, Relationship
between insecticidal and nerve activities of an imidacloprid-
related nitromethylene compound and its N-alkyl congeners.
Nihon Noyaku Gakkaishi ꢀJ Pestic Sci) 26:55±59 2001).
21 Nakayama A and Sukekawa M, Quantitative correlation between
molecular similarity and receptor-binding activity of neonico-
tinoid insecticides. Pestic Sci 52:104±110 1998).
6 Kodaka K, Kinoshita K, Wakita T, Yamada E, Kawahara N and
Yasui N, MTI-446: A novel systemic insect control com-
pound, in Proc 1998 Brighton Crop Protect Conf Pests and
Diseases, BCPC, Farnham, Surrey, UK, pp 21±26 1998).
7 Yamamoto I, Nicotine to nicotinoids: 1962 to 1997, in Nicotinoid
insecticides and the nicotinic acetylcholine receptor, ed by Yama-
moto I and Casida JE, Springer, Berlin, pp 3±27 1999).
8 Bai D, Lummis SCR, Leicht W, Breer H and Sattelle DB,
Actions of imidacloprid and a related nitromethylene on
cholinergic receptors of an identi®ed insect motor neuron.
Pestic Sci 33:197±204 1991).
22 Cramer RD III, Patterson DE and Bunce JD, Comparative
molecular ®eld analysis CoMFA). 1. Effect of shape on bind-
ing of steroids to carrier proteins. J Am Chem Soc 110:5959±
5967 1988).
23 Wold S, Ruhe A, Wold H and Dunn IIIWJ, The covariance
problem in linear regression. The partial least squares PLS)
approach in generalized inverses. SIAM J Sci Stat Comput
5:735±743 1984).
9 Nishimura K, Kanda Y, Okazawa A and Ueno T, Relationship
between insecticidal and neurophysiological activities of
imidacloprid and related compounds. Pestic Biochem Physiol
50:51±59 1994).
24 Kagabu S, Yokoyama K, Iwaya K and Tanaka M, Imidacloprid
and related compounds: Structure and water solubility of N-
alkylated derivatives of imidacloprid. Biosci Biotechnol Biochem
62:1216±1224 1998).
10 Matsuda K, Shimomura M, Kondo Y, Ihara M, Hashigami K,
Yoshida N, Raymond V, Mongan NP, Freeman JC, Komai K
25 Moriya K, Shibuya K, Hattori Y, Tsuboi S, Shiokawa K and
Kagabu S, 1- 6-Chloronicotinyl)-2-nitroimino-imidazolidines
and related compounds as potential new insecticides. Biosci
Biotech Biochem 56:364±365 1992).
and Sattelle DB, Role of loop
D of the a7 nicotinic
acetylcholine receptor in its interaction with the insecticide
imidacloprid and related neonicotinoids. Brit Pharmacol J
130:981±986 2000).
26 Nakagawa S, Okajima N, Kitahaba T, Nishimura K, Fujita T
and Nakajima M, Quantitative structure±activity studies of
substituted benzyl chrysanthemates. Pestic Biochem Physiol
17:243±258 1982).
11 Tomizawa M and Yamamoto I, Binding of nicotinoids and the
related compounds to the insect nicotinic acetylcholine
receptor. Nihon Noyaku Gakkaishi ꢀJ Pestic Sci) 17:231±236
1992).
27 Finney DJ, Probit analysis, Cambridge University Press, Cam-
bridge, UK 1952).
12 Liu M, Lanford J and Casida JE, Revalence of [3H]imidacloprid
binding site in house ¯y head acetylcholine receptor to
insecticidal activity of 2-nitromethylene- and 2-nitroimino-
imidazolidines. Pestic Biochem Physiol 46:200±206 1993).
13 Nishiwaki H, Nakagawa Y, Takeda DY, Okazawa Y, Akamatsu
M, Miyagawa H, Ueno T and Nishimura K, Binding activity of
substituted benzyl derivatives of chloronicotinyl insecticides to
house¯y-head membranes, and its relationship to insecticidal
activity against the house¯y Musca domestica. Pest Manag Sci
56:875±881 2000).
28 Sakuma M, Probit analysis of preference data. Appl Entomol Zool
33:339±347 1998).
29 Liu M, Lanford J and Casida JE, High af®nity binding of
[3H]imidacloprid in the insect acetylcholine receptor. Pestic
Biochem Physiol 46:40±46 1993).
30 Lind RJ, Clough MS, Reynolds SE and Earley FGP, [3H]imi-
dacloprid labels high- and low af®nity nicotinic acetylcholine
receptor-like binding sites in the aphid Myzus persicae
Hemiptera: Aphididae). Pestic Biochem Physiol 62:3±14
1998).
14 Nishiwaki H, Nakagawa Y, Ueno T, Kagabu S and Nishimura K,
Insecticidal and binding activities of N3-alkylated imidacloprid
derivatives against house¯ies Musca domestica and their a-
bungarotoxin binding sites of nicotinic acetylcholine receptors.
Pest Manag Sci 57:810±814 2001).
31 Nauen R, Ebbinghaus-Kintscher U, Elbert A, Jeschke P and
Tietjen K, Acetylcholine receptors as sites for developing
neonicotinoid insecticides, in Biochemical sites of insecticide
action and resistance, ed by Ishaaya I, Springer, Berlin, pp 77±
105 2000).
15 Nishiwaki H, Nakagawa Y, Ueno T and Nishimura K, Effects of
synergists on the insecticidal activity of chloronicotinyl-related
benzyl compounds against house¯ies. Nihon Noyaku Gakkaishi
ꢀJ Pestic Sci) 26:91±92 2001).
32 Nauen R, Ebbinghaus-Kintscher U and Schmuck R, Toxicity
and nicotinic acetylcholine receptor interaction of imidaclo-
prid and its metabolites in Apis mellifera Hymenoptera:
Apidae). Pest Manag Sci 57:577±586 2001).
16 Matsuo H, Tomizawa M and Yamamoto I, Structure±activity
relationships of acyclic nicotinoids and neonicotinoids for
insect nicotinic acetylcholine receptor/ion channel complex.
Arch Insect Biochem Physiol 37:17±23 1998).
33 Lansdell SJ and Miller NS, The in¯uence of nicotinic receptor
subunit composition upon agonist, a-bungarotoxin and imi-
dacloprid binding af®nity. Neuropharmacology 39:671±679
2000).
17 Okazawa A, Akamatsu M, Ohoka A, Nishiwaki H, Cho W-J,
Nakagawa Y, Nishimura K and Ueno T, Prediction of the
binding mode of imidacloprid and related compounds to
house-¯y head acetylcholine receptors using three-dimensional
QSAR analysis. Pestic Sci 54:134±144 1998).
34 Wollweber D and Tietjen K, Chloronicotinyl insecticides: a
success of the new chemistry, in Nicotinoid insecticides and the
nicotinic acetylcholine receptor, ed by Yamamoto I and Casida
JE, Springer, Berlin, pp 109±125 1999).
18 Okazawa A, Akamatsu M, Nishiwaki H, Nakagawa Y, Miyagawa
490
Pest Manag Sci 58:483±490 online: 2002)